Inherited human BCL10 deficiencies

Abstract

Human BCL10 deficiency causes combined immunodeficiency with bone marrow transplantation as its only curative option. To date, there are four homozygous mutations described in the literature that were identified in four unrelated patients. Here we describe a fifth patient with a novel mutation and summarize what we have learned about BCL10 deficiency. Due to the severity of the disease, accurate knowledge of its clinical and immunological characteristics is instrumental for early diagnosis and adequate clinical management of the patients.

INTRODUCTION

Caspase recruitment domain (CARD), B-cell lymphoma 10 (BCL10), and mucosa-associated lymphoid tissue lymphoma-translocation gene 1 (MALT1) proteins form the cytosolic CBM (CARD-BCL10-MALT1) complex. CARD family adaptor molecules are cell-type specific, with several known CARD molecules implicated that can form part of the CBM complex in a cell-type-specific: CARD-containing MAGUK protein 1 (CARMA1) (also known as CARD11) in lymphocytes, CARD9 in myeloid cells [1], CARMA3 (also known as CARD10) in epithelial cells [2-4] and CARD14 in keratinocytes [5]. In humans and mice, the CBM complex mediates NF-κB activation and mitogen-activated protein kinase (MAPK) activation signaling in a cell-type specific manner, nonredundant manner upon stimulation of several immunoreceptors. The NF-κB signaling pathway is critical in innate and adaptive immune regulation, cell memory, cell survival, and cell cycle progression. In lymphoid cells, the CBM complex controls lymphocyte activation and adaptive immunity by mediating T cell receptor (TCR) and B cell receptor (BCR) signaling [6-9]. In B cells, it is also involved in BAFF and TLR4 signaling [10,11], and in NK cells, it contributes to the signaling downstream of several killer cell receptors (NKRs), such as NK1.1, Ly49H, NKG2D, and Ly49D [12,13]. In myeloid cells, the CBM complex mediates innate immune responses by regulating NF-κB signaling downstream of immune receptors, including FcγR, Toll-like receptors (TLRs), C-type lectin receptors as dectin-1, dectin-2 and Mincle [14-21]. In epithelial cells, it has a role in TLR4 [14,22,23] and G protein–coupled receptor (GPCR) signaling [24,25]. Finally, in keratinocytes, CBM complex controls the expression of cytokines and chemokines in response to pathogen associated molecular patterns (PAMPs) and interleukin-17 (IL-17) [26].

BCL10 is a 233 amino acid intracellular signaling protein characterized by an amino-terminal CARD motif and a Ser/Thr-rich carboxyl terminus of unknown function [27,28]. BCL10 interacts with the two immunoglobulin (Ig)-like domains of MALT1 through a motif comprising a short stretch of amino acids following its CARD domain. Since 1999 it has been known that a genetic translocation of BCL10 is implicated in Mucosa-associated lymphoid tissue (MALT) lymphomas [29-31], especially in those with t(1;14)(p22;q32) or t(11;18)(q21;q21) chromosomal translocations. BCL10 is also a molecular determinant of other types of lymphoma [32-34]. In 2014, we described the first human BCL10 deficiency in a patient with a Combined Immunodeficiency (CID) [35]. Since then, we and others have reported three additional patients with BCL10 deficiency [36-38]. In this review, we describe a fifth patient with BCL10 deficiency that we recently identified and compile the genetic, immunological, and clinical features of all BCL10 deficient patients known so far.

BCL10 DEFICIENCY

Genetics and protein expression

All disease-causing BCL10 mutations described to date are homozygous, loss-of-function with a complete or significantly reduced protein expression (Table 1). A common feature of all the mutations is that they are located in the CARD domain of the molecule (Figure 1). The first reported mutation affected the first G nucleotide in intron 1, altering an essential splicing site (g.85741978C>T; IVS1+1G>A) (Table 1), resulting in aberrant splicing and, ultimately, the absence of the protein [35]. The subsequent two mutations were non-sense at positions K63X [36] and R88X [37], also causing a loss of expression (Table 1). The last two mutations are missense. The impact of the T91P on protein expression was not assessed [38], and the novel mutation R42H (Table 1, Figure 2A and 2B) that we describe in this report causes a considerable reduction of protein expression (Figure 2C). We speculate the slight protein expression is due to the forced overexpression system in the transfection of the mutant, which clearly must degrade rapidly because it is not stable. It is likely that no protein expression is seen in the patient's cells.

Table 1. Summary of human BCL10 deficiencies associated with CID.

MDDC: monocyte-derived dendritic cell; MDM: monocyte-derived macrophage; Hematopoietic stem cell transplantation: HSCT; Intravenous Immunoglobulin: IVIG.

Patients	Mutation /Protein expression	Consanguinity	Lymphocyte phenotype / Immunoglobulin levels	Cellular response	Main clinical features	Treatment
P1 [33]	g.85741978C>T (GRCh37.p13) IVS1+1G>A Homozygous. No protein expression.	Consanguineous parents	Normal total numbers of T and B cells but mostly with a naïve phenotype. Reduction of Tregs Hypogammaglobulinemia (IgG, IGA and IgM)	Normal responses to TLR1/2, TLR4, TLR2/6, and Dectin-1 signaling by MDMs and MDDCs. Impaired TLR4, TLR2/6, and Dectin-1 signaling in fibroblasts. Blocked T-cell proliferation in response to TCR stimulation.	6mo: Gastroenteritis, otitis and respiratory infections. 8mo: Viral infection (flu A and B, adenovirus; respiratory syncytial virus (RSV)); acute secondary respiratory failure, oral candidiasis and diaper dermatitis. 13mo: Prolonged diarrhea (Campylobacter Jejuni). 18mo: Diarrhea Active chronic colitis. 2y 5mo: Acute gastroenteritis (adenovirus) and respiratory infection (RSV). 2y 8mo: Diarrhea (adenovirus). Chronic non-spectfic colitis. 2y 10mo: Seizures and status epilepticus. Secondary diffuse leukoencephalopathy. 3y 4mo: Diarrhea (Clostridium difficile). Dizziness, disorientation and generalized weakness with focal abnormal movements. Suspected encephalitis. Died due to respiratory failure.	Deceased
P2 [35]	g.85270702G>A (GRCh38.p12) P.R88X Homozygous. No protein expression.	Consanguineous parents	Normal total numbers of T and B cells but mostly with a naïve phenotype. Hypogammaglobulinemia (IgG, IGA and IgM)	Impaired TLR4, TLR2/6, and Dectin-1 signaling in fibroblasts.	1mo: Flare of tie BCG scar with increased erythema and swelling 6mo: Severe viral lower respiratory tract infection and palatal ulcers. 8mo and 10mo: lower respiratory tract infections not requiring hospitalisation. 11mo: Acute onset respiratory distress (Mycobacterium tuberculosis and no evidence fungal infection).	HSCT
P3 [34]	g.85270779A>T (GRCh38.p12) P.K63X Homozygous, No protein expression.	Consanguineous parents	Lymphocitosis Absence of memory T and B cells. Reduction of NK, γδT, Tregs, and TFH cells. Hypogammaglobulinemia (IgM)		1yo: Hospitalized due to pneumonia (bacterial).	HSCT
P4 [36]	NM_003921.5: c.271A>C pT91P Homozygous, protein expression: not available	Consanguineous parents	Normal total numbers of T and low B cells numbers Hypogammaglobulinemia (IgG)	Blocked T-cell proliferation in response to TCR stimulation.	1mo: Scalp dermatitis with involvement of the trunk and axilla. Recurrent ear infections. Recurrent respiratory infections: Pneumocystis jirgyeci, adenovirus, acute respiratory distress syndrome (ARDS), bilateral pneumonia requiring ventilatory support. Unable to sit upright at 7mo.	HSCT (Deceased)
P5	g.85736522C>T (GRCh37.p13), P.R42H Homozygous, Low levels of protein expression.	Consanguineous parents	Normal total numbers of T and slight low B cells numbers Hypogammaglobulinemia (IgG and IgM)	Impaired TLR4, TLR2/6, and Dectin-1 signaling in fibroblasts.	Severe eczema; developmental delay; positive respiratory culture for Moraxella catarrhalis; pneumonia due to Klebsiella pneumoniae and skin infection due to Methicillin-resistant Staphylococcus aureus, Mollosum contagiousum and Micosporum canis.	Monthly IVIG and low potency steroid. No transplanted

Figure 1. Human BCL10 protein and mutations responsible of BCL10 deficiency.

Schematic representation of human BCL10 with its two domains and germline mutations known to cause BCL10 deficiency indicated. CARD: caspase recruitment domain; S/T rich: serine/threonine rich region.

Figure 2. A patient with CID and the novel BCL10 mutation: R42H.

(A) Familial segregation of the BCL10 mutation R42H. (B) Sanger sequencing results for the region encompassing the mutation for the patient (P5), the patient’s father (I1), and the patient’s mother (I2). (C) Immunoblot analysis of BCL10 protein in BCL10^−/−’s SV40-fibroblasts transfected with 2 μg of WT BCL10 plasmid (WT), empty vector (mock), BCL10 R42H plasmid (R42H), BCL10^−/− SV40-fibroblasts electroporated (E) or BCL10^−/− SV40-fibroblasts non-electroporated (NE). GAPDH was used as a loading control and to normalize the quantification of BCL10. The panels illustrate the results from a single experiment, representative of three. (D and E) Production of IL-6 and IL-8, respectively, as assessed by ELISA, in BCL10^−/− SV40 fibroblasts non-transfected (BCL10^−/−), BCL10^−/− SV40 fibroblasts transfected with 2 μg of WT BCL10 plasmid (WT), or R42H BCL10 plasmid (R42H) after stimulation 24 h post-transfection with poly(I:C), LPS, Zymosan, and Curdlan. Mean values ± SD were calculated from 3 independent experiments. Non-significant (ns), p < 0.05 (*), 0.005(**), <0.005-0.0001 (***), 0.0001 (****) for different stimuli between BCL10^−/− and WT or BCL10^−/− R42H.

The patients are all coming from consanguineous parents (Table 1). We showed in Torres et al. that T cell proliferation in the parents of the first patient was normal [35] and in García-Solís et al. that the frequencies of different lymphocyte subpopulations were normal in 5 heterozygous carriers [36]. Hence, we concluded that heterozygous carriers within the families are healthy and with normal immune cell frequencies suggesting that there is no haploinsufficiency in the BCL10 locus.

Immunology

The most significant immunological feature of BCL10 deficiency is that despite normal total numbers of T and B cells, their phenotype is naïve mainly with very low numbers of memory T and B cells. Only one patient (T91P) [38] showed B cell lymphocytopenia, with all patients displaying hypogammaglobulinemia (Table 1). In terms of cellular responses, the first patient analyzed revealed normal responses to TLR1/2, TLR4, TLR2/6, and Dectin-1 agonists by monocyte-derived macrophages (MDMs) or monocyte-derived dendritic cells (MDDCs). These findings highlighted that in the myeloid lineage, these immune receptors signal in a BCL10-independent manner. Interestingly, TLR4, TLR2/6, and Dectin-1 signaling in fibroblasts were impaired. T cell proliferation was measured in P1 and P4 and was blocked upon TCR stimulation (Table 1) [35,38]. We introduced the newly reported mutation R42H in BCL10-deficient fibroblasts and showed impaired IL6 and IL8 production in response to stimulation with TLR4, TLR2/6, and Dectin agonists, while TLR3 signaling is BCL10-independent (Figure 2D and 2E). These results indicate that consistent with other BCL10 mutations, R42H leads to a loss of function [35,37].

Clinical description and management

The first patient described showed the most complex clinical presentation. He was genetically diagnosed with BCL10 deficiency at three years of age, and he died before he could undergo hematopoietic stem cell transplantation (HSCT). Since six months of age, he suffered several infections at the respiratory and gut level: flu A and B, adenovirus; respiratory syncytial virus (RSV), gastroenteritis (adenovirus), prolonged diarrhea (Campylobacter jejuni), chronic colitis, as well as recurrent otitis, oral candidiasis and diaper dermatitis, secondary diffuse leukoencephalopathy, and encephalitis [35] (Table 1). Thanks to the description of this first patient, the rest of the patients’ diagnoses were fast, and HSCT was performed before their clinical state deteriorated. The second patient developed a flare of the BCG scar with increased erythema and swelling at one month of age. This episode was followed by several respiratory infections a few months later prior to genetic diagnosis and HSCT [37] (Table 1). The third patient was hospitalized due to bacterial pneumonia at one year old. Early genetic diagnosis allowed HSCT recommendation and treatment [36] (Table 1). The fourth patient had scalp dermatitis involving the trunk and axilla at one month of age. Some months later, she suffered respiratory and ear infections. After a genetic diagnosis, she was transplanted, but sadly she died [38] (Table 1). The fifth patient which we present for the first time in this review was 2 weeks of age at first presentation, now he is 4 years. He suffered severe eczema and developmental delay. Had a positive respiratory culture for Moraxella catarrhalis; he also suffered pneumonia due to Klebsiella pneumoniae and skin infection due to Methicillin-resistant Staphylococcus aureus, Mollosum contagiousum and Micosporum canis (Table 1). He did not need hospitalization since starting Intravenous Immunoglobulin (IVIG) and his eczema is controlled on a low potency steroid. He is not still transplanted.

CONCLUSIONS

In this review, we summarize the four previously published cases of CID caused by BCL10 deficiency and an additional one we just identified. Of the five mutations, their impact on protein expression was studied in four of them. Protein expression was absent for 3 of the mutations (IVS1+1G>A, R88X and K63X), and in one of them, it was greatly reduced (R42H) in overexpression system, speculating that it must be absent in physiological conditions. The mutations are always mapped to the CARD domain of BCL10, critical for protein-protein interactions and CBM complex assembly. BCL10 deficiency is an autosomal recessive trait with heterozygous carriers not showing clinical or immunological manifestations. The clinical hallmark of BCL10 deficiency is mainly respiratory infections since the first months of life, dermatitis, and gastrointestinal tract infections (Table 3). The majority of the patients had a sibling who died before the diagnosis of the index patient. In these patients, the only effective treatment is HSCT. Due to the severity of the disease, early genetic diagnosis is essential for prompt clinical management. This gene should be studied when a patient presents with recurrent infections, hypogammaglobulinemia, and reduced numbers of B and T memory lymphocytes. The current PID sequencing panels already include BCL10 in their list of genes. Still, when a candidate mutation appears disease-causing, performing a functional validation, including protein expression, is crucial to ensure that it is a BCL10 deficiency and thus proceed to HSCT.

Table 3. Summary of clinical manifestations of human BCL10 deficiencies.

Main clinical features
Respiratory infections:
∘ Viral infections: flu A and B, adenovirus; respiratory syncytial virus (RSV)
∘ Bacterial infections: Acute onset respiratory distress (Mycobacterium tuberculosis), Moraxella catarrhalis, pneumonia (Klebsiella pneumonia)
∘ Fungal infections: Pneumocystis jiroveci
Gastrointestinal manifestations:
∘ Gastroenteritis
∘ Prolonged diarrhea (Campylobacter jejuni)
∘ Acute gastroenteritis (adenovirus)
∘ Chronic non-specific colitis
∘ Diarrhea (Clostridium difficile)
Skin manifestations:
∘ Diaper dermatitis
∘ Scalp dermatitis with involvement of the trunk and axilla.
∘ Severe eczema
∘ Skin infection (Methicillin-resistant Staphylococcus aureus, Mollosum contagiousum and Micosporum canis)
Others manifestations:
∘ Otitis
∘ Oral candidiasis
∘ Secondary diffuse leukoencephalopathy
∘ Flare of the BCG scar with increased erythema and swelling
∘ Palatal ulcers
∘ Developmental delay

Table 2. Immune cells populations in peripheral blood and Immunoglobulin levels.

(A) Distribution of lymphocyte subpopulations in the peripheral blood of P5. (B) Immunoglobulin (Ig) levels (IgG, IgA and IgM) measured by nephelometry for P5.

A	P5 % of parental population	Healthy controls % of parental population [37,38]
CD19+	5	8-39
CD3+	91	56-75
CD3+CD4+	54	28-47
CD3+CD8+	36	16-30
CD16+CD56+	3	4-17
B
Immunoglobulin	Values g/L	Healthy controls g/L
IgG	0.62	2.95-11.56
IgA	0.31	0.27-2.46
IgM	0.18	0.37-2.24